Docosahexaenoic acid (DHA; cis-4,7,10,13,16,19-docosahexaenoic acid; C22:6 ω-3) is essential for the growth, functional development and healthy maintenance of brain function and is required throughout life from infancy through aging (Horrocks, L. A. and Y. K. Yeo. Pharmacol. Res. 40(3):211-225 (1999)). DHA deficiencies are associated with foetal alcohol syndrome, attention deficit hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility and adrenoleukodystrophy. In contrast, increased intake of DHA has been shown to be beneficial or have a positive effect in inflammatory disorders (e.g., rheumatoid arthritis), Type II diabetes, hypertension, atherosclerosis, depression, myocardial infarction, thrombosis, some cancers and for prevention of the onset of degenerative disorders such as Alzheimer's disease.
Fish (e.g., salmon, trout, mackerel) are an important source of DHA, since they naturally contain high concentrations of this long-chain fatty acid. Based on abundant research [reviewed in the 2005 Dietary Guidelines Advisory Committee Report for Americans, part D, section 4 (coordinated by the U.S. Dept. of Health & Human Services and the U.S. Dept. of Agriculture)], the American Heart Association, the National Cholesterol Education Program, the World Health Association, the European Society for Cardiology, the American Diabetes Association and the United Kingdom Scientific Advisory Committee on Nutrition all recommend two servings of fish per week (wherein each serving provides the equivalent of ˜450 mg per day of DHA and eicosapentaenoic acid (EPA, C20:5 ω-3)) for the cardioprotective effects so conveyed. As such, DHA is incorporated into a variety of products relating to functional foods, infant nutrition, bulk nutrition and animal health.
Although the physiological functions of docosapentaenoic acid (DPA, C22:5 ω-3) are still unknown, this fatty acid is the metabolic precursor of DHA and an immediate down-stream product of EPA via elongation. DPA is also known to be contained in fish oil, although the content is extremely low. The only known function for DPA is its usefulness as a carrier for transporting pharmaceutical agents into the brain (Japanese Patent Publication (Kokai) No. 61-204136 (1986)). It is expected, however, that DPA may play a physiological role in the animal body, since it is known that DPA increases in compensation for a lack of DHA (Homayoun et al., J. Neurochem., 51:45 (1988); Hamm et al., Biochem. J., 245:907 (1987); and Rebhung et al., Biosci. Biotech. Biochem., 58:314 (1994)). Thus, both DPA and DHA must be considered as important ω-3 fatty acids. One skilled in the art will recognize that the teachings herein that are directed toward DHA are also largely applicable and relevant to DPA production, should that become a desirable product in the future.
Although DHA is naturally found in different types of fish oil and marine plankton, it is expected that the supply of this ω-3 fatty acid will not be sufficient to meet growing demands. Fish oils have highly heterogeneous compositions (thereby requiring extensive purification to enrich for DHA), unpleasant tastes and odors (making removal economically difficult and rendering the oils unacceptable as food ingredients), and are subject to environmental bioaccumulation of heavy metal contaminants and fluctuations in availability (due to weather, disease and/or over-fishing).
As an alternative to fish oil, DHA can also be produced microbially. Generally, microbial oil production involves cultivating an appropriate microorganism that is naturally capable of synthesizing DHA in a suitable culture medium to allow for oil synthesis (which occurs in the ordinary course of cellular metabolism), followed by separation of the microorganism from the fermentation medium and treatment for recovery of the intracellular oil. Numerous different processes exist based on the specific microbial organism utilized [e.g., Schizochytrium species (U.S. Pat. Nos. 5,340,742; 6,582,941); Ulkenia (U.S. Pat. No. 6,509,178); Pseudomonas sp. YS-180 (U.S. Pat. No. 6,207,441); Thraustochytrium genus strain LFF1 (U.S. 2004/0161831 A1); Crypthecodinium cohnii (U.S. 2004/0072330 A1; de Swaaf, M. E. et al. Biotechnol Bioeng. 81(6):666-72 (2003) and Appl Microbiol Biotechnol. 61(1):40-3 (2003)); Emiliania sp. (Japanese Patent Publication (Kokai) No. 5-308978 (1993)); and Japonochytrium sp. (ATCC #28207; Japanese Patent Publication (Kokai) No. 199588/1989)]. Additionally, the following microorganisms are known to have the ability to produce DHA: Vibrio marinus (a bacterium isolated from the deep sea; ATCC #15381); the micro-algae Cyclotella cryptica and Isochrysis galbana; and, flagellate fungi such as Thraustochytrium aureum (ATCC #34304; Kendrick, Lipids, 27:15 (1992)) and the Thraustochytrium sp. designated as ATCC #28211, ATCC #20890 and ATCC #20891. And, athough several of these processes are not adaptable for industrial commercialization as a result of various limitations, there are at least three different fermentation processes for commercial production of DHA: fermentation of C. cohnii for production of DHASCO™ (Martek Biosciences Corporation, Columbia, Md.); fermentation of Schizochytrium sp. for production of an oil formerly known as DHAGold (Martek Biosciences Corporation); and fermentation of Ulkenia sp. for production of DHActive™ (Nutrinova, Frankfurt, Germany)). Despite these successes, each of these methods suffer from an inability to substantially improve the yield of oil or to control the characteristics of the oil composition produced, since the fermentations rely on the natural abilities of the microbes themselves.
Thus, microbial production of DHA using recombinant means is expected to have several advantages over production from natural microbial sources. For example, recombinant microbes having preferred characteristics for oil production can be used, since the naturally occurring microbial fatty acid profile of the host can be altered by the introduction of new biosynthetic pathways in the host and/or by the suppression of undesired pathways, thereby resulting in increased levels of production of desired PUFAs (or conjugated forms thereof) and decreased production of undesired PUFAs. Secondly, recombinant microbes can provide PUFAs in particular forms which may have specific uses. And, finally, microbial oil production can be manipulated by controlling culture conditions, notably by providing particular substrate sources for microbially expressed enzymes, or by addition of compounds/genetic engineering to suppress undesired biochemical pathways. Thus, for example, it is possible to modify the ratio of ω-3 to ω-6 fatty acids so produced, or engineer production of a specific PUFA (e.g., DHA) without significant accumulation of other PUFA downstream or upstream products.
Microbial production of DHA first requires the synthesis of the intermediate fatty acid, EPA. And, most microbially produced DHA is synthesized via the Δ6 desaturase/elongase pathway (which is predominantly found in higher plants, algae, mosses, fungi, nematodes and humans) and wherein: 1.) oleic acid is converted to LA by the action of a Δ12 desaturase; 2.) optionally, LA is converted to ALA by the action of a Δ15 desaturase; 3.) LA is converted to GLA, and/or ALA is converted to STA, by the action of a Δ6 desaturase; 3.) GLA is converted to DGLA, and/or STA is converted to ETA, by the action of a C18/20 elongase; 3.) DGLA is converted to ARA, and/or ETA is converted to EPA, by the action of a Δ5 desaturase; and 4.) optionally, ARA is converted to EPA by the action of a Δ17 desaturase (FIG. 1). However, an alternate Δ9 elongase/Δ8 desaturase pathway for the biosynthesis of EPA operates in some organisms, such as euglenoid species, where it is the dominant pathway for formation of C20 PUFAs (Wallis, J. G., and Browse, J. Arch. Biochem. Biophys. 365:307-316 (1999); WO 00/34439; and Qi, B. et al. FEBS Letters. 510:159-165 (2002)). In this pathway, 1.) LA and ALA are converted to EDA and ETrA, respectively, by a Δ9 elongase; 2.) EDA and ETrA are converted to DGLA and ETA, respectively, by a Δ8 desaturase; and 3.) DGLA and ETA are ultimately converted to EPA, as described above. Upon synthesis of EPA, a C20/22 elongase is responsible for conversion of the substrate to DPA, followed by desaturation by a Δ4 desaturase to yield DHA.
The literature reports a number of recent examples whereby various portions of the ω-3/ω-6 PUFA biosynthetic pathway have been introduced into Saccharomyces cerevisiae (a non-oleaginous yeast). Specifically, Dyer, J. M. et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)) reported synthesis of linolenic acids; Knutzon et al. (U.S. Pat. No. 6,136,574) demonstrated production of linoleic acid (LA), γ-linolenic acid (GLA), ALA and stearidonic acid (STA); Domergue, F. et al. (Eur. J. Biochem. 269:4105-4113 (2002)) described production of EPA; and Pereira, S. L. et al. (Biochem. J. 384:357-366 (2004)) were the first to produce DHA (3.8% of the total fatty acids, when fed EPA as substrate). Despite these successes, however, complex metabolic engineering has not been reported that would enable economical production of commercial quantities of DHA (i.e., greater than 5-30% with respect to total fatty acids). Additionally, considerable discrepancy exists concerning the most appropriate choice of host organism for such engineering.
Recently, Picataggio et al. (WO 2004/101757 and co-pending U.S. Patent Application No. 60/624,812) have explored the utility of oleaginous yeast, and specifically, Yarrowia lipolytica (formerly classified as Candida lipolytica), as a preferred class of microorganisms for production of PUFAs such as ARA, EPA and DHA. Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation can be up to about 80% of the cellular dry weight. Despite a natural deficiency in the production of ω-6 and ω-3 fatty acids in these organisms (since naturally produced PUFAs are limited to 18:2 fatty acids (and less commonly, 18:3 fatty acids)), Picataggio et al. (supra) have demonstrated production of 1.3% ARA and 1.9% EPA (of total fatty acids) in Y. lipolytica using relatively simple genetic engineering approaches and up to 28% EPA using more complex metabolic engineering. However, similar work has not been performed to enable economic, commercial production of DHA in this particular host organism.
Applicants have solved the stated problem by engineering various strains of Yarrowia lipolytica that are capable of producing greater than 5% DHA in the total oil fraction, using the Δ6 desaturase/Δ6 elongase pathway. Additional metabolic engineering and fermentation methods are provided to further enhance DHA productivity in this oleaginous yeast, as well as methodology to enable production of DHA via the Δ9 elongase/Δ8 desaturase pathway (thereby producing DHA-containing oil that is devoid of GLA).